Malaria remains a serious global health problem for which there is no effective vaccine. Previous studies indicate that animals can be immunized with inactivated sporozoites. The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Tissue culture cells infected with the recombinant synthesized polypeptides that reacted with monoclonal antibody against the malaria protein. Rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as malaria vaccines.